Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. In recent studies, the effect of DNA-tension on DNA-protein interaction has been demonstrated. Here, we study single-molecule DNA hydrolysis by exonucleaseIII (exoIII) which has 3''''-≫ 5'''' exonuclease activity. We observed the exoIII digestion of individual stretched DNA molecules from the free end. The shortening of fluorescently stained DNA was recorded and analyzed. The sequentially captured photographs demonstrate that the digested DNA molecule linearly shortened with reaction time. We also carried out the single-molecule observation without buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the relaxed condition. The correlation between physical form of DNA and digestion rate of exoIII was clearly demonstrated by single-molecule observations.
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